Electrochemiluminescence (ECL) has received widespread attention during the previous decade, especially in the field of chemical analysis. It combines the well known sensitivity of chemiluminescence (CL) with the precise control over the time and position of light emitting reactions afforded by electrochemistry. As an alternative approach for conducting immunoassays and nucleotide assays it offers advantages such as increased sensitivity and precision, reduction in time and labor, and the elimination of radioisotopes. In order to exploit the full potential of this technology there is a requirement for new chemiluminescent compounds which can be initiated electrochemically. We show for the first time how CL can be triggered by electrochemical oxidation of acridan compounds.
Prior to the present invention, ruthenium chelates and luminol derivatives were the only compounds that have been used in a significant number of analytical applications involving ECL. (J. K. Leland and M. J. Powell, J. Electrochem. Soc., 1990, 137, 3127; S. Sakura, Anal. Chim. Acta., 1992, 262, 49) Ruthenium chelates have been used for enzyme assays, but their most significant impact has been as labels for immunoassays and nucleotide assays. In these applications a combination of ECL and magnetic bead technology has found increasing use in pharmaceutical labs for high throughput screening. Luminol has also been used for enzyme assays and immunoassays. Light is emitted when electrochemically oxidized luminol reacts with hydrogen peroxide which allows the reaction to be coupled to oxidase enzymes such as glucose oxidase. (R. Wilson and A. P. F. Turner, Biosensors, 1997, 12, 277) The chemiluminescence reaction of luminol is also catalyzed by electrochemically oxidized ferrocenes (R. Wilson and D. J. Schiffrin, J. Electroanal. Chem., 1998, 448, 125) suggesting that these compounds could be used as labels in an ECL system resembling the one based on ruthenium chelates.
Acridinium esters were discovered in 1964 and subsequently developed as labels for immunoassays and nucleotide assays. The chemiluminescence reaction mechanism of these compounds involves nucleophilic attack of a peroxide anion (HOO—) in alkaline solution on the 9-position of the acridinium nucleus followed by internal cyclization leading to the formation of a metastable dioxetanone intermediate. This spontaneously decarboxylates to give the singlet excited state of N-methylacridone, which emits blue light at 430 nm when it relaxes to the ground state. The chemiluminescence quantum yield is typically between 1 and 10%. The reaction is extremely rapid, but in the absence of peroxide other nucleophiles such as hydroxide ion can form an adduct (pseudo-base) with the 9-position of the acridinium nucleus. Formation of this intermediate precludes the formation of a dioxetanone intermediate and therefore no light is emitted unless pseudo-base formation is reversed by an acidic solution of hydrogen peroxide before adding a sodium hydroxide solution.
Electrochemical triggering of the chemiluminescent reaction of an acridinium ester at a pH of 5.0 was developed in an attempt to simplify the conventional initiation procedure. (J. S. Littig and T. A. Neeman, Anal. Chem., 1992, 64, 1140–1144) This pH is not particularly useful for immunoassays and nucleotide assays. A solution of acridinium ester was injected into a flowing stream of pH 12 phosphate buffer and pumped into a flow cell. Chemiluminescence was triggered in the cell by reducing dissolved oxygen electrochemically. The conditions are a compromise between those required for chemiluminescence and oxygen reduction, and those necessary to avoid pseudo-base formation. It would also be necessary to control the concentration of dissolved oxygen to obtain precise results which cancels out the increase in simplicity obtained by initiating the chemiluminescent reaction electrochemically. These drawbacks are avoided when an acridan ester is used because the acridinium ester is produced in situ from a passive precursor.
Recently a large number of acridans (reduced acridinium esters, thioesters and amides) based on the N-alkylacridancarboxylate nucleus, including DMC, have been made. (H. Akhavan-Tafti, et al., J. Org. Chem. 1998, 63, 930–937; H. Akhavan-Tafti et al., Clin. Chem. 1995, 41, 1368–1369) These acridan compounds are stable in the presence of hydrogen peroxide and do not form an inactive pseudo-base. Light emission can be triggered by enzymatically oxidizing the acridan with the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide and an enhancer such as p-iodophenol. HRP oxidizes the acridan to the corresponding acridinium ester, which in most cases is immediately subject to nucleophilic attack by the peroxide anion (HOO—) at the 9 position of the acridinium nucleus; the possibility of pseudo-base formation does not arise because peroxide is several orders of magnitude more nucleophilic than hydroxide. Nucleophilic attack on the acridinium ester results in the formation of a dioxetanone which decomposes to form the singlet excited state of N-methylacridone. This in turn relaxes to the ground state accompanied by the emission of intense blue light with a maximum wavelength of 430 nm. By using these compounds as a substrate for HRP it has been possible to detect as little as 0.1 amol of this enzyme in a 15 minute assay.
Previous work on the electrochemistry of acridan which does not bear a carbonyl group at the 9-position demonstrated the oxidation by a mechanism in which the second oxidation step occurs in solution as a result of disproportionation between protonated and unprotonated radical intermediates. (P. Hapiot, J. Moiroux and J. M. Saveant, J. Am. Chem. Soc., 1990, 112, 1337) This reaction did not involve the production of chemiluminescence.
Acridan compounds substituted with an oxidizable exocyclic double bond are disclosed in commonly assigned U.S. Pat. No. 5,922,558. These compounds are enzymatically oxidized by a peroxidase enzyme to produce visible light. The opposite terminus of the double bond bears two substituents, one being an ether or thioether-type group, the other being any of various groups such as ether or thioether-type groups, alkoxy, aryloxy, alkylthio, arylthio, trialkylsilyloxy, phosphoryloxy, acyloxy and acylthio groups. Compounds of this type having a phosphate salt group are also disclosed in commonly assigned U.S. Pat. No. 6,045,727 which describes their enzymatic reaction with phosphatase enzymes to produce chemiluminescence.